Development of a dose-limiting data collection strategy for serial synchrotron rotation crystallography

Area of Science:

• Structural biology
• X-ray crystallography

Background:

• Serial crystallography, including serial femtosecond crystallography (SFX), shows promise for protein microcrystallography.
• Serial synchrotron crystallography (SSX) is explored as an alternative, but faces challenges with radiation damage due to longer exposure times.

Purpose of the Study:

• To evaluate the advantages of sample rotation in serial synchrotron crystallography (SSX).
• To investigate the influence of radiation dose on data quality and mercury single-wavelength anomalous diffraction (Hg-SAD) phasing.
• To determine optimal rotation parameters for collecting high-quality diffraction data and phasing using Hg-SAD.

Main Methods:

• Serial synchrotron rotation crystallography was employed.
• Mercury single-wavelength anomalous diffraction (Hg-SAD) phasing was performed on luciferin regenerating enzyme (LRE) microcrystals.
• The impact of varying rotation steps and radiation doses on data quality, resolution, and anomalous signal was analyzed.

Main Results:

• Sample rotation proved effective for accurate data collection in SSX.
• The optimal helical rotation step varied depending on factors like multiplicity, partiality, exposure time, and background scattering.
• For LRE microcrystals, a 0.25° rotation step maximized resolution, while steps ≥1° were better for Hg-SAD phasing.
• Increased resolution and anomalous signal were observed up to 3.4 MGy due to improved signal-to-noise, despite specific damage at the Hg site beyond 1.1 MGy.

Conclusions:

• Sample rotation is a crucial technique for enhancing data collection in serial synchrotron crystallography.
• Careful selection of rotation step and radiation dose is necessary to balance data quality, resolution, and phasing capabilities.
• SSX with Hg-SAD phasing offers a viable approach for determining protein structures from microcrystals.